The AAA ATPases family includes molecules whose roles include, but are far from limited to, biogenesis of mitochondria and multivesicular bodies, proteins in involved in gene regulation and protein transport. This project will focus on studying the activity of these machines at the single molecule level. The model protein will be ClpX - it is the part of the proteosome, a key effect of protein degradation that unfolds the proteins to prepare them for degradation. . Cellular proteins differ widely in their liability, from half-lives of minutes to days. Regulated degradation, by allowing rapid changes in the levels of cellular proteins, helps control signal transduction pathways, the cell- cycle, transcription, apoptosis, antigen processing, biological clock control, differentiation and surface receptor desensitization. The questions to be addressed are: How is work partitioned between alternative outcomes? What is the maximum work that can be performed by the system? What factors limit its efficiency in performing work? These questions have health implications: human pathological conditions are associated with failures of the degradation system and its regulation offers the potential for therapeutic intervention. Furthermore, an inhibitor of proteasome catalytic activity is in use for treatment of recurrent multiple myeloma, and proteasome inhibitors are in clinical trial for treatment of a broad spectrum of human malignancies. Thus, understanding the regulation of the half-life of proteins should provide critical insights into cell physiology and pathology. The mishandling of aberrant proteins incurs penalties throughout biology: the survival of bacteria subjected to stress depends on the effective performance of systems which deal with misfolded and structurally aberrant proteins- to either fold them properly or destroy them. The specific questions to be addressed are: How hard can the device pull to cause unfolding? How many pulls are needed to commit irreversibly? What is the limit of pulling power, and the statistical distribution of pulling power? Answers to these questions will begin to reveal not just what these machines do but the decision tree that describes how outcomes are controlled and when machine capacity may be exceeded. We want to know not just how the machine works, but how its decision tree yields alternative outcomes.